Multi-layer airway organoids and methods of making and using the same

ABSTRACT

Provided herein are artificial lung organoids. The artificial lung organoids may include an epithelial cell layer comprising mammalian lung epithelial cells, a stromal cell layer comprising mammalian lung fibroblast cells and an endothelial cell layer comprising mammalian endothelial cells. The artificial lung organoids may optionally include a porous membrane between said epithelial cell layer and said stromal cell layer and/or between said stromal cell layer and said endothelial lung cell layer.

RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalPatent Application Ser. Nos. 62/242,611, filed Oct. 16, 2015, and62/404,931, filed Oct. 6, 2016, the contents of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates generally to multi-layer airway organoids andmethods of making and using of the same.

BACKGROUND OF THE INVENTION

The study of respiratory infection is significantly limited by a lack ofsuitable in vivo and in vitro models to investigate interactions betweenthe respiratory epithelium, infection and disease. For example, animalmodels often do not acquire the pathological abnormalities in theairways and lungs seen in humans. See, e.g., Guilbault et al., Cysticfibrosis mouse models, American Journal of Respiratory Cell andMolecular Biology. 2007; 36(1):1-7. Additionally, most in vitro modelsare unable to create the differentiated tissue components and structuralcomplexity of the airway epithelium. See, e.g., Lang et al.,Three-dimensional culture of hepatocytes on porcine liver tissue-derivedextracellular matrix, Biomaterials. 2011; 32(29):7042-7052.

Primary airway epithelial cells, derived from cadaver tissue andexpanded ex vivo on 2D plastic culture surfaces, remain the currentstandard for disease modeling and therapy evaluation in vitro. However,these techniques present cells with artificial conditions, includingtwo-dimensional (2D) growth surfaces that are several magnitudes stifferthan most soft tissues, and lack of important signals from the tissuemicroenvironment. As a consequence, they impose a selective pressure onthe cells that substantially alter their heterogeneity and functionalproperties. See, e.g., Anderson et al., Tumor morphology and phenotypicevolution driven by selective pressure from the microenvironment, Cell127.5 (2006): 905-915. For example, plastic culture expanded cells oftenbecome non-ciliated, a significant limitation in studying bacterialpathogens of the airways, which often display preferential attachment tociliated respiratory epithelium in vivo. See, e.g., Matsui et al.,Evidence for periciliary liquid layer depletion, not abnormal ioncomposition, in the pathogenesis of cystic fibrosis airways disease.Cell. 1998; 95(7):1005-1015; Gray et al., Mucociliary differentiation ofserially passaged normal human tracheobronchial epithelial cells.American Journal of Respiratory Cell and Molecular Biology. 1996;14(1):104-112. The lack of physiological airway models represents asignificant limitation to the study of the pathogenesis of infection inthe airway.

US Patent Application Publication 2009/0227025 to Nichols et al.discussed the use of progenitor or stem cells to generate new lungtissue in an in vitro system using microgravity conditions. U.S. Pat.No. 8,647,837 to Mahmood et al. and U.S. Pat. No. 5,750,329 to Quinn etal. discuss the use of alveolar and endothelial cell layers in anartificial tissue construct for the study of lung diseases concerningthe alveoli, or air sacs, of the lungs. U.S. Pat. No. 8,338,114 toGoodwin discusses three-dimensional (3D) human broncho-epithelialtissue-like assemblies produced in a rotating wall vessel withmicrocarriers by co-culturing mesenchymal bronchial-tracheal cells andbronchial epithelium cells. However, there remains a need for improvedin vitro systems that can be used for study of infection andpathogenesis affecting the lungs.

SUMMARY OF THE INVENTION

Provided herein is an artificial mammalian lung organoid, comprising:

(a) an epithelial cell layer comprising mammalian lung epithelial cells;

(b) a stromal cell layer comprising mammalian lung fibroblast cells; and

(c) an endothelial cell layer comprising mammalian endothelial cells(e.g., microvascular endothelial cells).

In some embodiments, the organoid further comprises a porous membrane(e.g., a polymeric material) between said epithelial cell layer and saidstromal lung cell layer and/or between said stromal lung cell layer andsaid endothelial lung cell layer.

In some embodiments, the cells of the lung epithelial cell layer arepolarized. In some embodiments, the cells of the lung endothelial celllayer, stromal cell layer and/or epithelial cell layer are human.

In some embodiments, the lung organoid is an upper airway lung organoid.In some embodiments, the mammalian lung epithelial cells are bronchialepithelial cells. In some embodiments, the mammalian lung epithelialcells comprise normal bronchial epithelial cells. In some embodiments,the mammalian lung epithelial cells comprise diseased bronchialepithelial cells.

In some embodiments, the bronchial epithelial cells comprise basal,goblet, ciliated and/or clara cells.

In some embodiments, the ratio of mammalian lung fibroblast cells of thestromal layer and mammalian endothelial cells of the endothelial celllayer is from 2:1 to 1:2. In some embodiments, the ratio of mammalianlung fibroblast cells of the stromal layer and mammalian epithelialcells of the epithelial cell layer is from 2:1 to 1:2. In someembodiments, the ratio of mammalian endothelial cells of the endotheliallayer and mammalian epithelial cells of the epithelial cell layer isfrom 2:1 to 1:2.

In some embodiments, the porous membrane is coated on one or both sideswith laminin, collagen type I, collagen type IV, fibronectin, elastin, alung tissue-derived extracellular matrix composition, or a combinationthereof.

In some embodiments, the lung organoid is infected with a lung pathogen.In some embodiments, the lung organoid is infected with Bordetellapertussis or Pseudomonas aeruginosa.

Also provided is a microfluidic device comprising the lung organoid astaught herein. The microfluidic device may include a housing comprisinga chamber and a channel The lung organoid may be in the chamber, thechannel may be configured to provide fluid connection of the lungorganoid to a fluid inlet and a fluid outlet, and said fluid inlet andsaid fluid outlet may be connected to a fluid reservoir.

In some embodiments, at least a portion of the housing may betransparent. In some embodiments, the endothelial cell layer of the lungorganoid is in fluid connection with a liquid (e.g., media) in thedevice. In some embodiments, the epithelial cell layer of the lungorganoid is in fluid connection with a gas (e.g., air). In someembodiments, the epithelial cell layer of the lung organoid is in fluidconnection with a liquid (e.g., media).

Methods of making the lung organoid as taught herein are also provided.According to some embodiments, the method may include:

depositing the endothelial cell layer comprising the mammalianendothelial cells onto a first side of the porous membrane;

depositing the stromal cell layer comprising the mammalian lungfibroblast cells onto a second side of the porous membrane that isopposite the first side of the porous membrane; and

depositing the epithelial cell layer comprising the mammalian lungepithelial cells directly onto the stromal cell layer.

In some embodiments, the method may include:

providing a hydrogel including the mammalian lung fibroblast cells;

depositing the endothelial cell layer comprising the mammalianendothelial cells onto a first side of the hydrogel; and

depositing the epithelial cell layer comprising the mammalian lungepithelial cells onto an a second side of the hydrogel that is oppositethe first side of the hydrogel.

In some embodiments, the hydrogel may be cross-linked. In someembodiments, the hydrogel may include gelatin, fibrinogen, gellan gum,pluronics (poloxamers), alginate, chitosan, hyaluronic acid, celluloseand/or collagen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 (a), (b) and (c) provide schematic illustrations of the layersof lung organoids including membrane(s) according to some embodiments ofthe present invention.

FIG. 2 shows a 3D lung organoid developed by layering fluorescentdye-labeled microvasculature endothelial cells and airway stromalmesenchymal cells with primary airway epithelium.

FIG. 3 provides a schematic illustration of a lung organoid including ahydrogel according to some embodiments of the present invention.

FIG. 4 (a) is a schematic diagram of a microfluidic device according tosome embodiments of the present invention; FIG. 4 (b) is a schematicdiagram of a microfluidic support system according to some embodimentsof the present invention; and FIG. 4 (c) is an image of a microfluidicsupport system according to some embodiments of the present invention.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention is now described more fully hereinafter. Thisinvention may, however, be embodied in many different forms and shouldnot be construed as limited to the embodiments set forth herein; ratherthese embodiments are provided so that this disclosure will be thoroughand complete and will fully convey the scope of the invention to thoseskilled in the art.

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a,” “an” and “the” are intended toinclude plural forms as well, unless the context clearly indicatesotherwise. It will be further understood that the terms “comprises” or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements components and/or groups orcombinations thereof, but do not preclude the presence or addition ofone or more other features, steps, operations, elements, componentsand/or groups or combinations thereof.

As used herein, the term “and/or” includes any and all possiblecombinations or one or more of the associated listed items, as well asthe lack of combinations when interpreted in the alternative (“or”).

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the specification andclaims and should not be interpreted in an idealized or overly formalsense unless expressly so defined herein. Well-known functions orconstructions may not be described in detail for brevity and/or clarity.

“Cells” as used herein are, in general, mammalian cells, such as dog,cat, cow, goat, horse, sheep, mouse, rabbit, rat, etc. cells. In somepreferred embodiments the cells are human cells. Suitable cells areknown and are commercially available, and/or may be produced inaccordance with known techniques. In some embodiments, the cells areharvested from a donor and passaged. In some embodiments, the cells aredifferentiated from cell lines. In some embodiments, the cells arederived from adult stem cells (bone marrow, peripheral blood, umbilicalcord blood, wharton's jelly in the umbilical cord or from placentaltissues), embryonic stem cells, amniotic fluid stem cells, or any othersource of stem cells that can be differentiated into the tissue ofinterest.

“Mammalian” as used herein refers to both human subjects (and cellssources) and non-human subjects (and cell sources or types), such asdog, cat, mouse, monkey, etc. (e.g., for veterinary purposes).

“Extracellular Matrix” (ECM) as used herein refers to extracellularmolecules secreted by cells that provides structural and biochemicalsupport to the surrounding cells. The ECM is normally composed of aninterlocking mesh of fibrous proteins and polysaccharides such asglycosaminoglycans (GAGs). “Extracellular Matrix composition” as usedherein refers to a composition including ECM proteins.

“Extracellular Matrix Proteins” (or “ECM proteins”) as used herein areknown, and include but are not limited to those described in Y. Zhang etal., US Patent Application Publication No. 2013/0288375. Examples of ECMproteins include, but are not limited to, laminin, collagen type I,collagen type IV, fibronectin and elastin.

“Organoid” as used herein refers to an artificial, in vitro constructcreated to mimic or resemble the functionality and/or histologicalstructure of an organ or portion thereof.

“Media” or “culture media” as used herein refers to an aqueous basedsolution that is provided for the growth, viability, or storage of cellsused in carrying out the present invention. A media or culture media maybe natural or artificial. A media or culture media may include a basemedia and may be supplemented with nutrients (e.g., salts, amino acids,vitamins, trace elements, antioxidants) to promote the desired cellularactivity, such as cell viability, growth, proliferation, and/ordifferentiation of the cells cultured in the media. A “base media,” asused herein, refers to a basal salt nutrient or an aqueous solution ofsalts and other elements that provide cells with water and certain bulkinorganic ions essential for normal cell metabolism and maintainsintra-cellular and/or extra-cellular osmotic balance. In someembodiments, a base media may include at least one carbohydrate as anenergy source and/or a buffering system to maintain the medium withinthe physiological pH range. Examples of commercially available basemedia may include, but are not limited to, phosphate buffered saline(PBS), Dulbecco's Modified Eagle's Medium (DMEM), Minimal EssentialMedium (MEM), Basal Medium Eagle (BME), Roswell Park Memorial InstituteMedium (RPMI) 1640, MCDB 131, Click's medium, McCoy's 5A Medium, Medium199, William's Medium E, insect media such as Grace's medium, Ham'sNutrient mixture F-10 (Ham's F-10), Ham's F-12, α-Minimal EssentialMedium (αMEM), Glasgow's Minimal Essential Medium (G-MEM) and Iscove'sModified Dulbecco's Medium. See, e.g., US Patent Application PublicationNo. US20150175956.

“Hydrogel” as used herein refers to naturally-derived hydrogels andsynthetic hydrogels. Naturally-derived hydrogels and synthetic hydrogelsmay be mixed to form hybrid hydrogels. Naturally-derived hydrogels mayinclude, but not limited to, Matrigel®, which is made out of nativeextracellular matrix proteins collected from a cell line, collagen andalginate. Naturally-derived hydrogels may be derived from decellularizedtissue extracts. Extracellular matrix may be collected from a specifictissue and may be used as or combined with a hydrogel material to beused to support cells of that tissue type. See, e.g., Skardal et al.,Tissue Specific Synthetic ECM Hydrogels for 3-D in vitro Maintenance ofHepatocyte Function, Biomaterials 33 (18): 4565-75 (2012). Chitosanhydrogel is an example of a naturally-derived hydrogel that isdegradable and supportive for several different cell types. See, e.g.,Moura et al., In Situ Forming Chitosan Hydrogels Prepared viaIonic/Covalent Co-Cross-Linking, Biomacromolecules 12 (9): 3275-84(2011). Hyaluronic acid hydrogels may also be used. See, e.g., Skardalet al., A hydrogel bioink toolkit for mimicking native tissuebiochemical and mechanical properties in bioprinted tissue constructs,Acta Biomater. 25: 24-34 (2015).

Synthetic hydrogels may be produced from a variety of materials (e.g.,Poly-(ethylene glycol)) and using many techniques. In contrast tonaturally-derived hydrogels, synthetic hydrogels may be produceduniformly and may be easily reproducible and characterized. Synthetichydrogels may, however, lack some functional signals for cells, like theactive sites found in natural extracellular matrix, limiting theirpotential to support cells. See, e.g., Mahoney et al., Three-DimensionalGrowth and Function of Neural Tissue in Degradable Polyethylene GlycolHydrogels, Biomaterials 27 (10): 2265-74 (2006). Hybrid hydrogels mayoffer a compromise and may allow for more control over the ability toreconstruct a specific microenvironment. By combining naturalcomponents, such as extracellular matrix molecules (e.g., extracellularmatrix proteins), with defined synthetic hydrogels, a more easilyreproducible and functional hydrogels can be produced. See, e.g.,Salinas et al., Chondrogenic Differentiation Potential of HumanMesenchymal Stem Cells Photoencapsulated within Poly(EthyleneGlycol)—Arginine-Glycine-Aspartic Acid-Serine Thiol-MethacrylateMixed-Mode Networks, Tissue Engineering 13 (5): 1025-34 (2007).

The disclosures of all United States patent references cited herein areto be incorporated by reference to the extent that they are consistentwith the disclosures herein.

1. Lung Organoids and Methods of Making the Same

Provided herein is an artificial mammalian lung organoid including (a)an epithelial cell layer including mammalian lung epithelial cells, (b)a stromal cell layer including mammalian lung fibroblast cells, and (c)an endothelial cell layer including mammalian endothelial cells. In someembodiments, the epithelial cell layer may include primary mammalianlung epithelial cells. In some embodiments, the stromal cell layer mayinclude primary mammalian lung fibroblast cells or mammalian lungfibroblast cells differentiated from a stem cell or cell line, and theendothelial cell layer may include primary mammalian endothelial cellsor mammalian endothelial cells differentiated from a stem cell or cellline. In some embodiments, the endothelial cell layer may also includemicrovascular endothelial cells. In some embodiments, the endothelialcell layer may include human umbilical vein endothelial cells (HUVEC).

In some embodiments, said mammalian lung epithelial cells, saidmammalian lung fibroblast cells and/or said mammalian endothelial cellsmay be human cells. In some embodiments, the mammalian lung epithelialcells are polarized cells that include distinct “apical”, “lateral” and“basal” plasma membrane domains. In some embodiments, polarizedepithelial cells allow directional transport of molecules across theepithelial layer.

In some embodiments, the lung organoid may further include a membranebetween said epithelial cell layer and said stromal lung cell layerand/or between said stromal lung cell layer and said endothelial lungcell layer.

FIGS. 1 (a), (b) and (c) provide schematic illustrations of the layersof lung organoids including membrane(s) according to some embodiments ofthe present invention. The lung organoid may include a membrane 10between the epithelial cell layer 20 and the stromal lung cell layer 30as illustrated in FIG. 1(a), may include a membrane 10 between thestromal lung cell layer 30 and the endothelial lung cell layer 40 asillustrated in FIG. 1(b) or may include a membranes 10 between theepithelial cell layer 20 and the stromal lung cell layer 30, and betweenthe stromal lung cell layer 30 and the endothelial lung cell layer 40,respectively, as illustrated in FIG. 1(c). In some embodiments, themembrane(s) included in the lung organoid may be porous, allowing theflow or transport of molecules through them.

In some embodiments, lung organoids of the invention may be made by:

depositing an endothelial cell layer comprising live mammalianendothelial cells (e.g., microvascular endothelial cells) on a firstside of a membrane (e.g., porous membrane);

depositing a stromal cell layer comprising live mammalian lungfibroblast cells on a second side of the membrane that is opposite thefirst side of the membrane; and

depositing an epithelial cell layer comprising live mammalian lungepithelial cells directly on the stromal cell layer.

Cells may be obtained from established cultures, donors, biopsy,immortalized cell lines, stem cells, or a combination thereof. In someembodiments, cells are primary cells. In some embodiments, cells arehuman lung cells. In some embodiments, cells are passaged.

Depositing or seeding of the cells can be carried out by any suitabletechnique, including but not limited to spreading/painting, coating,spraying, etc. In some embodiments the depositing steps may be carriedout by printing or bioprinting in accordance with any suitabletechnique, including “ink jet” type printing, syringe injection typeprinting or other methodology known in the art. Apparatus for carryingout such bioprinting is known and described in, for example, Boland etal., U.S. Pat. No. 7,051,654; Yoo et al., US Patent Application Pub. No.US 2009/0208466; and Kang et al., US Patent Application Publication No.US 2012/0089238.

As noted above, a membrane (e.g., a porous membrane) may be positionedat one or more junctions of the cell layers of the lung organoid. Themembrane may be or include a polymeric material. The polymeric materialmay be synthetic, such as polystyrene, or derived from a natural tissue,such as a decelluarized extracellular matrix (ECM) or non-decelluarizedextracellular matrix (ECM). In some embodiments, one or both sides ofthe membrane may be coated with ECM protein (e.g., laminin, collagentype I, collagen type IV, fibronectin and elastin), proteoglycan,vitronectin, poly-D-lysine and/or polysaccharide.

In some embodiments, one or both sides of the membrane may be coated onwith a lung tissue-derived extracellular matrix composition or ahydrogel including a lung tissue-derived extracellular matrixcomposition. See, e.g., Lang et al., Three-dimensional culture ofhepatocytes on porcine liver tissue-derived extracellular matrix,Biomaterials. 2011; 32(29):7042-7052; Mirmalek-Sani et al., Porcinepancreas extracellular matrix as a platform for endocrine pancreasbioengineering, Biomaterials. 2013; 34(22):5488-5495; Orlando et al.,Production and implantation of renal extracellular matrix scaffolds fromporcine kidneys as a platform for renal bioengineering investigations,Annals of surgery. 2012; 256(2):363-370; see also Booth et al.,Acellular normal and fibrotic human lung matrices as a culture systemfor in vitro investigation, American Journal of Respiratory and CriticalCare Medicine. 2012; 186(9):866-876. For example, the lung ECM biogelmay be generated by solubilizing human lung ECM powder and combining thesolubilized human lung ECM powder with a hydrogel. The human lung ECMpowder may be made from decellularized or non-decelluarized human lungECM. The human lung ECM powder may be formed by lyophilizing human lungECM and then grinding the lyophilized human lung ECM into a powder,e.g., with a freezer mill. See e.g., Y. Zhang et al., US PatentApplication Publication No. 2013/0288375; and Skardal et al., Tissuespecific synthetic ECM hydrogels for 3-D in vitro maintenance ofhepatocyte function, Biomaterials 33(18): 4565-75 (2012).

In some embodiments, lung organoids of the invention may be made by:

providing a hydrogel including stromal cells (e.g., live mammalian lungfibroblast cells) therein;

depositing an endothelial cell layer comprising live mammalianendothelial cells (e.g., microvascular endothelial cells) on a firstside of the hydrogel; and

depositing an epithelial cell layer comprising live mammalian lungepithelial cells on a second side of the hydrogel that is opposite thefirst side of the hydrogel.

In some embodiments, the hydrogel may or may not be cross-linked. Insome embodiments, the hydrogel may include gelatin, fibrinogen, gellangum, pluronics (poloxamers), alginate, chitosan, hyaluronic acid,cellulose and/or collagen.

In some embodiments, 3D lung organoids may be formed by layeringmicrovasculature endothelial cells (lower) and airway stromalmesenchymal cells (middle) with primary airway epithelium (upper) asillustrated in FIG. 2. The epithelial cells are polarized and ciliated.In some embodiments, endothelial cells may be labeled with fluorescentdye.

FIG. 3 provides a schematic illustration of a lung organoid including ahydrogel according to some embodiments of the present invention.Referring to FIG. 3, in some embodiments, the lung organoid may furtherinclude a hydrogel 50 that includes said stromal cells 30 therein. Theepithelial cell layer 20 and endothelial cell layer 40 may be onopposing surfaces of the hydrogel 50, respectively. The hydrogel 50 mayor may not be cross-linked. Examples of the hydrogel 50 include, but arenot limited to, gelatin, fibrinogen, gellan gum, pluronics (poloxamers),alginate, chitosan, hyaluronic acid, cellulose, collagen, and mixturesthereof.

In some embodiments, the hydrogel 50 may include a lung tissue-derivedextracellular matrix composition (a lung ECM biogel). In someembodiments, the stromal cells of the organoid may be suspended in thelung ECM biogel at physiological stiffness (1-2 kPa). In someembodiments, the hydrogel 50 may be a hyaluronic acid-based hydrogel.See e.g., Skardal et al., A hydrogel bioink toolkit for mimicking nativetissue biochemical and mechanical properties in bioprinted tissueconstructs, Acta Biomater. 25: 24-34 (2015); and PCT ApplicationPublication No. WO 2016/064648A1, which is incorporated by referenceherein.

In some embodiments, the lung organoid may be infected by a viral orbacterial lung pathogen. Pathogens that cause respiratory diseaseinclude the common flu or influenza (A or B; Orthomyxoviridae family),respiratory syncytial virus, human parainfluenza viruses (HPIVs;paramyxovirus family), metapneumovirus (hMPV; family Paramyxoviridae),adenoviruses, rhinoviruses, parainfluenza viruses, coronaviruses,coxsackievirus, and herpes simplex virus. Respiratory disease bacterialpathogens include Yersinia pestis, Bacillus anthracis, Escherichia coli,Pseudomonas aeruginosa, Francisella tularensis, Staphylococcus aureusGroup A beta-hemolytic streptococci (GABHS), group C beta-hemolyticstreptococci, Corynebacterium diphtheriae, Neisseria gonorrhoeae,Arcanobacterium haemolyticum, Chlamydia pneumoniae, Mycoplasmapneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxellacatarrhalis, Bordetella pertussis, and Bordetella parapertussis.

2. Devices and Systems

In some embodiments, the lung organoid may be provided in a microfluidicdevice. Various microfluidic device configurations useful for thesupport of organoids are known in the art. See, e.g., US PatentApplication Publication No. 2014/0038279 to Ingber et al.; Bhatia andIngber, “Microfluidic organs-on-chips,” Nature Biotechnology 32:760-772(2014).

In general, a microfluidic device including the lung organoid as taughtherein may include a chamber so dimensioned to accept the lung organoidtherein such that the lung organoid defines a boundary between a firstchamber or opening in fluid contact with the epithelial cell layer ofthe lung organoid, and a second chamber or opening in fluid contact withthe endothelial cell layer of the lung organoid. The fluid may be aliquid such as media, or a gas such as air. The device may furtherinclude a fluid inlet and fluid outlet for each chamber, fluidreservoirs connected therewith, etc. In some embodiments, a gas (e.g.,air) may contact the epithelial cell layer of the lung organoid, and aliquid (e.g., media) may contact the endothelial cell layer of the lungorganoid. In some embodiments, a microfluidic device may be provided inthe form of a cartridge for “plug in” or insertion into a largerapparatus including pumps, culture media reservoir(s), detectors, andthe like.

FIG. 4 (a) is a schematic diagram of a microfluidic device according tosome embodiments of the present invention. Referring to FIG. 4(a), insome embodiments, a microfluidic device may include a housing 510 and achamber 520 in the housing 510. A lung organoid 530 may be placed in thechamber 520. The microfluidic device may further include a fluid inletand fluid outlet 540 through which fluid (e.g., media, air) may come inand out and a channel or an opening 550 which are provided for fluidconnection of the lung organoid 530 to the fluid. In some embodiments,at least a portion of the microfluidic device housing 510 may betransparent to allow imaging the lung organoid 530. In some embodiments,the microfluidic device may include multiple fluid inlets and outlets540 and multiple channels 550 for providing different types of fluids tothe lung organoid 530. In some embodiments, electrodes of atrans-endothelial (or epithelial) electrical resistance (TEER) sensormay be provided in the housing 510 of the microfluidic device (notshown).

FIG. 4 (b) is a schematic diagram of a microfluidic support systemaccording to some embodiments of the present invention. Referring toFIG. 4(b), the microfluidic support system may include one or more of acontroller 100 for pump control (e.g., a computer), a pump 200 (e.g., aperistaltic pump), a bubble trap 300, a media reservoir 400 and amicrofluidic device 500. It will be understood that the microfluidicsupport system may include multiple reservoirs 400 for different typesof media or other fluids.

FIG. 4 (c) is an image of a microfluidic support system according tosome embodiments of the present invention. The microfluidic supportsystem includes two media reservoirs that have a cylindrical shape andare connected to a pump through tubing and a microfluidic device thathas a rectangular shape and is connected to both the media reservoirsand the pump through the tubing to form a closed-loop system.

3. Methods of Use

The lung organoids as described herein may be used as an alternative tolive animal testing for compound or vaccine screening (e.g., screeningfor efficacy, toxicity, or other metabolic or physiological activity) orfor treatment of (including resistance to treatment of) lung infectionor disease (e.g., chronic obstructive pulmonary disease (COPD)). Foracute treatment testing, compound or vaccine may be applied, e.g., oncefor several hours. For chronic treatment testing, compound or vaccinemay be applied, e.g., for days to one week. Such testing may be carriedout by providing a lung organoid product as described herein underconditions which maintain constituent cells of that product alive (e.g.,in a culture media with oxygenation); applying a compound to be tested(e.g., a drug candidate) to the lung organoid (e.g., by topical or vaporapplication to the epithelial layer); and then detecting a physiologicalresponse (e.g., damage, scar tissue formation, infection, cellproliferation, burn, cell death, marker release such as histaminerelease, cytokine release, changes in gene expression, etc.), thepresence of such a physiological response indicating said compound orvaccine has therapeutic efficacy, toxicity, or other metabolic orphysiological activity if inhaled or otherwise delivered into the lungof a mammalian subject. A control sample of the lung organoid may bemaintained under like conditions, to which a control compound (e.g.,physiological saline, compound vehicle or carrier) may be applied, sothat a comparative result is achieved, or damage can be determined basedon comparison to historic data, or comparison to data obtained byapplication of dilute levels of the test compound, etc.

It will be understood that the lung organoids as described herein can bean excellent tool to study drug delivery since the lung organoidsinclude both an epithelial cell layer and an endothelial cell layer. Theendothelial cell layer of the lung organoids may be exposed to a liquid(e.g., media) and may function as a mature vascular barrier thatcontrols materials passing through the endothelial cell layer. Theepithelial cell layer of the lung organoids may be exposed to a gas(e.g., air) and thus may be exposed to materials delivered by aerosol.In some embodiment, the epithelial cell layer may include cilia.

Methods of determining whether a test compound has immunologicalactivity may include testing for immunoglobulin generation, chemokinegeneration and cytokine generation by the cells.

4. Storing and Shipping of Devices

Once produced, the devices described above may be used immediately, orprepared for storage and/or transport.

To store and transport the device, a transient protective support mediathat is a flowable liquid at room temperature (e.g., 25° C.), but gelsor solidifies at refrigerated temperatures (e.g., 4° C.), such as agelatin mixed with water, may be added into the device to substantiallyor completely fill the chamber(s), and preferably also any associatedconduits. Any inlet and outlet ports may be capped with a suitablecapping element (e.g., a plug) or capping material (e.g., wax). Thedevice may be then packaged together with a cooling element (e.g., ice,dry ice, a thermoelectric chiller, etc.) and all may be placed in a(preferably insulated) package.

In some embodiments, to store and transport the device, a transientprotective support media that is a flowable liquid at cooled temperature(e.g., 4° C.), but gels or solidifies at warm temperatures such as roomtemperature (e.g., 20° C.) or body temperature (e.g., 37° C.), such aspoly(N-isopropylacrylamide) and poly(ethylene glycol) block co-polymers,may be added into the device to substantially or completely fill thechamber(s), and preferably also any associated conduits.

Upon receipt, the end user may simply remove the device from theassociated package and cooling element, may allow the temperature torise or fall (depending on the choice of transient protective supportmedia), may uncap any ports, and may remove the transient protectivesupport media with a syringe (e.g., by flushing with growth media).

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPLES

An airway organoid was constructed using primary normal human bronchialepithelial (NHBE) cells, primary human lung fibroblasts and humanendothelial cells (HUVEC), as found in the normal human upper airway,layered on a polyester membrane in an order and ratio to replicate humanairway tissue.

1. Construction of Lung Organoid

Each side of a polyester membrane (Corning® Costar® Snapwell™ cellculture inserts, 12 mm with 0.4 μm pore, pore density 4×106 pores/cm²,polyester membrane, TC-treated, sterile) was coated with 150 μl collagenIV (Sigma C7521) and left under the biosafety hood overnight (hood openand blower on). The membrane was UV sterilized for 30 mins the nextmorning.

250,000 HUVECs (endothelial cells) (Cell and Viral Vector CoreLaboratory, Wake Forest University) were seeded on the underside of themembrane and let stand for up to 4 hours for cells to attach. Membranewas then placed with 2 ml of EGM-10 in the well of the 6 well plate.

250,000 HALF (human adult lung fibroblasts, isolated from donor) wereseeded on the upper side of the membrane and covered with 200 μl ofDMEM-F12 1:1 (Hyclone SH30261.01).

After 5-7 days, a layer of 250,000 cells NHBE cells (epithelial cells)(Lonza Cat #CC2540) was established over the layer of fibroblasts andcovered with 200 μl BEGM (Clonetics CC4175).

Change media: EGM 10 and BEGM—Once every 2 days; DMEM—Once every 3-4days by carefully pipetting off 100 μl media from the top of thetranswell (for HALF& NHBE) without touching the cells and replacing itwith the same amount of fresh media.

Primary cell types were characterized by PCR and flow cytometry todemonstrate normal cell phenotype using Vybrant® MulticolorCell-Labeling Kit V-22889 (Used for labeling cells for imaging). p63-α(D2K8X) XP® Rabbit mAb #13109, Anti-Dynein intermediate chain 1 antibody[74.1] (ab23905) and Anti-Mucin 5AC antibody [45M1] (ab3649) were usedfor characterization of NHBE by flow-cytometry.

A microfluidic device was used to provide physiological flow of media toboth sides of the membrane, or alternatively, to the lower side only,producing an air-liquid interface established at the upper membrane.

Pathogenesis of Bordetella pertussis was studied in this model byanalyzing trans-epithelial resistance, the levels of toxins andcytokines, imaging by fluorescent microscopy.

2. Results and Discussion

Characterization of cells: Primary lung fibroblasts maintainedexpression of fibroblast-specific markers during passaging, while NHBEcells maintained expression of markers for basal, goblet, ciliated andclara cells during culture.

Normal human lung fibroblasts are tested for expression of vonWillebrand factor/Factor VIII, cytokeratins 18 and 19, and alpha smoothmuscle actin. Lung microvascular endothelial cells express CD31/105, vonWillebrand Factor, and PECAM. Human Primary lung Epithelial cellsexpress markers basal (p63+ KRT5+), ciliated (Foxj1+ Sox17+), Clara(Scgb1A1+) mucosal (Muc5ac+) cell numbers and CFTR+ epithelial cells.

Organoid Development: Fluorescent labeling of the cells of the airwayorganoid demonstrated that the cells formed distinct cell layersrepresentative of endothelial vasculature, the stromal component and apolarized epithelial monolayer. TEM and histological imaging confirmedthe development of a multi-layered upper airway organoid construct.

Maintenance of cells in microfluidic system: The airway organoidmaintained in the microfluidic system remained viable and facilitatednon-invasive analysis of Bordetella pertussis pathogenesis.

Conclusion

Multiple primary airway cell types were combined to generate afunctional upper airway organoid. This bioengineered organoid system isuseful in conducting studies into human disease, toxicity studies anddrug and vaccine development.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. An artificial mammalian lung organoid, comprising: (a) an epithelialcell layer comprising mammalian lung epithelial cells; (b) a stromalcell layer comprising mammalian lung fibroblast cells; and (c) anendothelial cell layer comprising mammalian endothelial cells (e.g.,microvascular endothelial cells), optionally wherein said organoidfurther comprises a porous membrane between said epithelial cell layerand said stromal cell layer and/or between said stromal cell layer andsaid endothelial cell layer.
 2. The lung organoid of claim 1, whereinthe mammalian lung epithelial cells are polarized.
 3. The lung organoidof claim 1, wherein the mammalian endothelial cells are human cells. 4.The lung organoid of claim 1, wherein the mammalian lung fibroblastcells are human cells.
 5. The lung organoid of claim 1, wherein themammalian lung epithelial cells are human cells.
 6. The lung organoid ofclaim 1, wherein said organoid is an upper airway lung organoid.
 7. Thelung organoid of claim 1, wherein the mammalian lung epithelial cellsare bronchial epithelial cells.
 8. The lung organoid of claim 1, whereinthe mammalian lung epithelial cells comprise normal bronchial epithelialcells.
 9. The lung organoid of claim 1, wherein the mammalian lungepithelial cells comprise diseased bronchial epithelial cells.
 10. Thelung organoid of claim 8, wherein the bronchial epithelial cellscomprise basal, goblet, ciliated and/or clara cells.
 11. The lungorganoid of claim 1, wherein a ratio of the mammalian lung fibroblastcells of the stromal cell layer and the mammalian endothelial cells ofthe endothelial cell layer is from 2:1 to 1:2.
 12. The lung organoid ofclaim 1, wherein a ratio of the mammalian lung fibroblast cells of thestromal cell layer and the mammalian lung epithelial cells of theepithelial cell layer is from 2:1 to 1:2.
 13. The lung organoid of claim1, wherein said porous membrane comprises a polymeric material.
 14. Thelung organoid of claim 1, wherein at least one side of said porousmembrane is coated with an extracellular matrix composition.
 15. Thelung organoid of claim 14, wherein said extracellular matrix compositionis collagen.
 16. The lung organoid of claim 1, wherein saidextracellular matrix composition is a lung tissue-derived extracellularmatrix composition.
 17. The lung organoid of claim 16, wherein said lungtissue-derived extracellular matrix composition is derived from adecellualized lung extracellular matrix.
 18. The lung organoid of claim1, further comprising a hydrogel, wherein said stromal cell layer is insaid hydrogel.
 19. The lung organoid of claim 18, wherein said hydrogelcomprises gelatin, fibrinogen, gellan gum, pluronics (poloxamers),alginate, chitosan, hyaluronic acid, cellulose and/or collagen.
 20. Thelung organoid of claim 18, wherein said hydrogel comprises anextracellular matrix composition.
 21. The lung organoid of claim 20,wherein said extracellular matrix composition comprises a lungextracellular matrix composition.
 22. The lung organoid of claim 1,wherein said organoid is infected with a lung pathogen.
 23. The lungorganoid of claim 1, wherein said organoid is infected with Bordetellapertussis or Pseudomonas aeruginosa.
 24. A microfluidic devicecomprising the lung organoid of claim 1, the device comprising a housingcomprising a chamber and a channel, wherein the lung organoid is in thechamber, wherein the channel is configured to provide fluid connectionof the lung organoid to a fluid inlet and a fluid outlet, and whereinsaid fluid inlet and said fluid outlet are connected to a fluidreservoir.
 25. The microfluidic device of claim 24, wherein at least aportion of the housing is transparent.
 26. The microfluidic device ofclaim 24, wherein the endothelial cell layer of the lung organoid is influid connection with a liquid.
 27. The microfluidic device of claim 24,wherein the epithelial cell layer of the lung organoid is in fluidconnection with a gas.
 28. The microfluidic device of claim 24, whereinthe epithelial cell layer of the lung organoid is in fluid connectionwith a liquid.
 29. A method of making the lung organoid of claim 1, themethod comprising: depositing the endothelial cell layer comprising themammalian endothelial cells onto a first side of the porous membrane;depositing the stromal cell layer comprising the mammalian lungfibroblast cells onto a second side of the porous membrane that isopposite the first side of the porous membrane; and depositing theepithelial cell layer comprising the mammalian lung epithelial cellsdirectly onto the stromal cell layer.
 30. The method of claim 29,wherein said depositing steps are carried out by spreading, painting,coating, printing, bioprinting, and/or spraying.
 31. A method of makingthe lung organoid of claim 1, the method comprising: providing ahydrogel including the mammalian lung fibroblast cells; depositing theendothelial cell layer comprising the mammalian endothelial cells onto afirst side of the hydrogel; and depositing the epithelial cell layercomprising the mammalian lung epithelial cells onto an a second side ofthe hydrogel that is opposite the first side of the hydrogel.
 32. Themethod of claim 31, wherein said depositing steps are carried out byspreading, painting, coating, printing, bioprinting, and/or spraying.33. The method of claim 31, wherein the hydrogel is cross-linked. 34.The method of claim 31, wherein the hydrogel comprises gelatin,fibrinogen, gellan gum, pluronics (poloxamers), alginate, chitosan,hyaluronic acid, cellulose and/or collagen.